【作者】 杨丽娜；

【导师】 林俊芳；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2003， 硕士

【摘要】 本研究利用cDNA-AFLP改良法分离了13个草菇菌丝低温诱导表达基因v1，v2，v3，v4，v5，v6，v7，v8，v9，v10，v11，v12，v13。DNA序列测定结果表明，V1为488bp，V2为551bp，V3为557bp，V4为285bp，V5为173bp，V6为604bp，V7为743bp，V8为836bp，V9为709bp，V10大约为720bp（未测完），V11和V12是同一基因片段为1077bp，V13为617bp。BLASTn搜索结果表明V1，V2，V3，V4，V5，V6，V7，V9，V10，V11，V12，V13为未知新基因，V8与ATP酶同源率较高，在一段长312bp的区域，与ATP酶的同源率达84-93％。BLASTx搜索结果表明：V1，V2，V3，V4，V6，V8，V11（V12），V13的mRNA翻译产物能够找到同源率较高的蛋白，其中六个基因片段，V1，V2，V3，V4，V8，V11的mRNA翻译产物与其他真菌蛋白的同源率较高。V3的mRNA翻译产物与灰盖鬼伞细胞色素P450的同源率为33％，V8的mRNA翻译产物与粗糙脉胞菌的ca²⁺-ATP酶同源性为68％。 本研究以草菇基因组DNA为模板，设计了一对引物（rasR1，rasF1），利用PCR技术克隆了草菇的一段启动子片段r5a。DNA序列测定结果及DNA序列对比结果显示：该启动子序列（r5a）与已知的食用菌ras启动子同源率低，采用Plant Prediction和Plant Care软件对克隆的草菇启动子片段进行分析，结果表明该启动子序列的243-293bp及539-589bp区域为预测的基础启动子区，有两个转录起始位点（283bp及579bp处），有多种重要的顺式作用元件，如TATA-box，CAAT-box，G-box，ABRE元件，WUN-motif，HSE元件，P-box，TATC-Vbox，I-box等。更多还原

【Abstract】 Using a single restriction enzyme based on cONA-AFLP to isolate the differential gene, we got 13 differentially expressed gene fragments, we named them, Vl(488bp), V2(551bp), V3(557bp), V4(285bp), V5(173bp), V6(604bp), V7(743bp), V8(836bp), V9(709bp), V10(about720bp), V11(1077bp), V12(l077bp), V13(617bp). Vll and V12 are the same gene fragments. Using BLASTn to analyse these sequences, we got the following results: VI, V2, V3, V4, V5, V6, V7, V9, V10, 11(V12), V13 are the unknown gene. V8 is 84-93% the same as ATPase gene in a region of 312bp. Using BLASTx, we know that: V1, V2, V3, V4, V11(V12), V13 are more than 30% the same as the protein of other fungus. V3 is about 33% the same as cytochrome P450(from Coprinopsis cinerea ), V8 is about 68% the same as ca2+-ATPase (from Neurospora crassa).Using PCR technology to isolate ras promoter, we got a DMA sequence that is likely to be a promoter sequence. This DNA sequence is not the same as other ras promoter sequences in edible mushroom. Two possible foundational promoter sequences locate on the region of 243-293bp and 539-589bp.There are two transcription start points. One locate at the site of 283bp,another locate at the site of 579bp. There are also many important cis-acting regulatory elements, such as TATA-box, CMT-box, G-box, ABRE, WUN-motif, HSE, P-box, TATC-box, I-box et al.更多还原